U.S. patent application number 15/421889 was filed with the patent office on 2017-08-03 for spherical microcapsules with enhanced oral bioavailability.
This patent application is currently assigned to I2O Pharma Ltd.. The applicant listed for this patent is I2O Pharma Ltd.. Invention is credited to Michel Afargan, Dan Gelvan, Natalia Naraykin.
Application Number | 20170216217 15/421889 |
Document ID | / |
Family ID | 59386329 |
Filed Date | 2017-08-03 |
United States Patent
Application |
20170216217 |
Kind Code |
A1 |
Afargan; Michel ; et
al. |
August 3, 2017 |
SPHERICAL MICROCAPSULES WITH ENHANCED ORAL BIOAVAILABILITY
Abstract
In one embodiment, the present invention provides a composition
comprising a plurality of microcapsules, each comprising a shell
and a core carrying an active agent selected from the group
consisting of: (a) a non-hydrophilic active agent and (b) a
hydrophilic active agent dissolved or suspended in an oil; wherein
the shell comprises a polymeric coating, and wherein at least a
portion of the microcapsules in a sample of the composition are
spherical when the sample of the microcapsules is viewed in a
scanning electron microscope with a magnification in the range of
between .times.2000 and .times.50000.
Inventors: |
Afargan; Michel; (Ramat Gan,
IL) ; Gelvan; Dan; (Ramat Gan, IL) ; Naraykin;
Natalia; (Ramat Gan, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
I2O Pharma Ltd. |
Ramat Gan |
|
IL |
|
|
Assignee: |
I2O Pharma Ltd.
Ramat Gan
IL
|
Family ID: |
59386329 |
Appl. No.: |
15/421889 |
Filed: |
February 1, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62289526 |
Feb 1, 2016 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/5026 20130101;
Y02A 50/30 20180101; A61K 38/03 20130101; A61K 9/5192 20130101;
A61K 9/5123 20130101; A61K 38/31 20130101; A61K 9/5047 20130101;
A61K 31/513 20130101; Y02A 50/411 20180101; A61K 9/5089 20130101;
A61K 47/26 20130101; A61K 38/26 20130101; A61K 9/5153 20130101 |
International
Class: |
A61K 9/50 20060101
A61K009/50; A61K 47/26 20060101 A61K047/26; A61K 38/03 20060101
A61K038/03; A61K 9/51 20060101 A61K009/51; A61K 31/513 20060101
A61K031/513 |
Claims
1. A composition comprising: a plurality of microcapsules, each
comprising a shell and a core carrying an active agent selected
from the group consisting of: (a) a non-hydrophilic active agent
and (b) a hydrophilic active agent dissolved or suspended in an
oil; wherein the shell comprises a polymeric coating, and wherein
at least a portion of the microcapsules in a sample of the
composition are spherical when the sample of the microcapsules is
viewed in a scanning electron microscope with a magnification in
the range of between .times.2000 and .times.50000.
2. The composition of claim 1, wherein the non-hydrophilic active
agent is a lipophilic agent.
3. The composition of claim 1, wherein the active agent is a
hydrophilic agent with a molecular weight of about 1 kiloDalton or
about 4 kiloDalton.
4. The composition of claim 1, further comprising a plurality of
nanocapsules situated within a lumen formed by each shell of the
plurality of microcapsules.
5. The composition of claim 4, wherein the plurality of
nanocapsules are accommodated in a gel forming polymer.
6. The composition of claim 5, wherein the plurality of
nanocapsules each comprise a shell comprising a polymeric coating
and a liquid oil core comprising an active agent dissolved or
suspended in the liquid oil core.
7. A method of preparing a spherical microcapsules comprising a
plurality of nanocapsules, the method comprising: (a) spray drying
sugar in a spray drying evaporator comprising a spray chamber and a
cyclone, the amount of sugar being sufficiently in excess to cause
at least a portion of the sugar to adhere to the surfaces of the
evaporator; (b) spray drying a dispersion comprising an active
agent encapsulated in oil core droplets of the plurality of
nanocapsules and a gel forming polymer or a combination of gel
forming polymers to obtain microcapsules, wherein the active agent
is selected from the group consisting of: (a) a non-hydrophilic
active agent and (b) a hydrophilic active agent dissolved or
suspended in an oil; wherein a shell of the oil core droplets
comprises a polymeric coating, and wherein at least a portion of
the microcapsules are spherical when viewed in a scanning electron
microscope with a magnification in the range of between .times.2000
and .times.50000.
8. A method of preparing spherical microcapsules comprising a
plurality of nanocapsules situated within a lumen formed by a shell
of the spherical microcapsule, the nanocapsules comprising an oil
core carrying an active agent and a polymeric shell coating, the
method comprising: (a) spray drying sugar in a spray drying
evaporator, the amount of sugar being sufficiently in excess to
cause at least a portion of the sugar to adhere to the interior
collection surfaces of the evaporator; (b) providing an organic
phase comprising oil, a water miscible organic solvent, an active
agent dissolved in the solvent and a polymer or combination of
polymers for coating the oil core, wherein the active agent is
selected from the group consisting of: (a) a non-hydrophilic active
agent and (b) a hydrophilic active agent dissolved or suspended in
an oil; (c) slowly adding water to the organic phase to obtain an
emulsion; (d) continuously adding water to the emulsion to induce
phase inversion of the emulsion thereby obtaining an oil in water
(o/w) emulsion; (e) mixing the o/w emulsion with a gel forming
polymer or a combination of gel forming polymers; and (f) removing
the organic solvent by means of evaporation and water by means of
spray drying to obtain the spherical microcapsules.
9. A pharmaceutical dosage form comprising: a composition and a
physiologically acceptable carrier, wherein the composition
comprises a plurality of microcapsules, each comprising a shell and
a core carrying an active agent selected from the group consisting
of: (a) a non-hydrophilic active agent and (b) a hydrophilic active
agent dissolved or suspended in an oil; wherein the shell comprises
a polymeric coating, and wherein at least a portion of the
microcapsules in a sample of the composition are spherical when the
sample of the microcapsules is viewed in a scanning electron
microscope with a magnification in the range of between .times.2000
and .times.50000.
10. The pharmaceutical dosage form of claim 9, further comprising a
plurality of nanocapsules situated within a lumen formed by each
shell of the plurality of microcapsules.
11. The pharmaceutical dosage form of claim 10, wherein the
plurality of nanocapsules each comprising comprise a shell of
comprising a polymeric coating and a liquid oil core comprising an
active agent active agent dissolved or suspended in the liquid oil
core.
12. The pharmaceutical dosage form of claim 9, wherein the
physiologically acceptable carrier is suitable for oral
administration or suitable for administration by injection.
13. A method of increasing bioavailability of an active agent in a
human subject's body; the method comprising: administering to the
human subject a composition or a pharmaceutical dosage form
comprising the composition and a physiologically acceptable
carrier, wherein the composition comprises a plurality of
microcapsules, each comprising a shell and a core carrying an
active agent selected from the group consisting of: (a) a
non-hydrophilic active agent and (b) a hydrophilic active agent
dissolved or suspended in an oil; wherein the shell comprises a
polymeric coating, and wherein at least a portion of the
microcapsules in a sample of the composition are spherical when the
sample of the microcapsules is viewed in a scanning electron
microscope with a magnification in the range of between .times.2000
and .times.50000.
14. The method of claim 13, wherein the delivery of the active
agent is via the intestinal paracellular, trancellular or lymphatic
transport, or a combination thereof.
15. The method of claim 13, wherein the physiologically acceptable
carrier is suitable for oral administration or suitable for
administration by injection.
16. A method of treating a subject for a pathological condition
which requires for the treatment an effective blood level of an
active agent, the method comprising: administering to the subject a
composition or a pharmaceutical dosage form comprising the
composition and a physiologically acceptable carrier, wherein the
composition comprises a plurality of microcapsules, each comprising
a shell and a core carrying an active agent selected from the group
consisting of: (a) a non-hydrophilic active agent and (b) a
hydrophilic active agent dissolved or suspended in an oil; wherein
the shell comprises a polymeric coating, and wherein at least a
portion of the microcapsules in a sample of the composition are
spherical when the sample of the microcapsules is viewed in a
scanning electron microscope with a magnification in the range of
between .times.2000 and .times.50000.
17. The method of claim 16, wherein the physiologically acceptable
carrier is suitable for oral administration or suitable for
administration by injection.
18. The method of claim 16, wherein the delivery of the active
agent is via the intestinal paracellular, trancellular or lymphatic
transport, or a combination thereof.
Description
CROSS-REFERENCE
[0001] This application claims priority to U.S. Provisional Patent
Application Ser. No. 62/289,526, filed on Feb. 1, 2016, the entire
content of which is incorporated by reference in its entirety.
[0002] The present invention relates to microcapsules for oral
delivery.
BACKGROUND
[0003] Oral administration of drugs is considered to be a
convenient route of administration. However, certain drugs have
poor bioavailability when administered orally. Encapsulation of
drugs into microparticles can improve the bioavailability of drugs
administered orally.
SUMMARY
[0004] In one embodiment, the present invention provides a
composition comprising a plurality of microcapsules, each
comprising a shell and a core carrying an active agent selected
from the group consisting of: (a) a non-hydrophilic active agent
and (b) a hydrophilic active agent dissolved or suspended in an
oil; wherein the shell comprises a polymeric coating, and wherein
at least a portion of microcapsules in a sample of the composition
are spherical when the sample of the microcapsules is viewed in a
scanning electron microscope with a magnification in the range of
between .times.2000 and .times.50000.
[0005] In one embodiment, the non-hydrophilic active agent is a
lipophilic agent. In one embodiment, the non-hydrophilic active
agent is lopinavir. In one embodiment, the active agent is a
hydrophilic agent with a molecular weight of about 1 kiloDalton
(kD). In one embodiment, the hydrophilic agent with a molecular
weight of about 1 kiloDalton (kD) is octreotide. In one embodiment,
the active agent is a hydrophilic agent with a molecular weight of
about 4 kiloDalton (kD). In one embodiment, the hydrophilic agent
with a molecular weight of about 4 kiloDalton (kD) is
exenatide.
[0006] In one embodiment, the present invention provides a
composition comprising a plurality of nanocapsules situated within
a lumen formed by each shell of the plurality of microcapsules. In
one embodiment, the plurality of nanocapsules are accommodated in
the gel forming polymer, the plurality of nanocapsules each
comprising a shell of polymeric coating and a liquid oil core
comprising an active agent dissolved or suspended in the liquid oil
core.
[0007] In one embodiment, the present invention provides a method
of preparing a spherical microcapsules comprising a plurality of
nanocapsules, the method comprises: [0008] (a) spray drying sugar
in a spray drying evaporator comprising a spray chamber and a
cyclone, the amount of sugar being sufficiently in excess to cause
at least a portion of the sugar to adhere to the surfaces of the
evaporator; [0009] (b) spray drying a dispersion comprising an
active agent encapsulated in oil core droplets of the plurality of
nanocapsules and a gel forming polymer or a combination of gel
forming polymers to thereby obtain microcapsules, wherein the
active agent is selected from the group consisting of: (a) a
non-hydrophilic active agent and (b) a hydrophilic active agent
dissolved or suspended in an oil; [0010] wherein a shell of the oil
core droplets comprises a polymeric coating, and; wherein at least
a portion of the microcapsules are spherical when viewed in a
scanning electron microscope with a magnification in the range of
between .times.2000 and .times.50000.
[0011] In one embodiment, the present invention provides a method
of preparing spherical microcapsules comprising a plurality of
nanocapsules situated within a lumen formed by a shell of the
spherical microcapsule, the nanocapsules comprising an oil core
carrying an active agent and a polymeric shell coating, the method
comprising: (a) spray drying sugar in a spray drying evaporator,
the amount of sugar being sufficiently in excess to cause at least
a portion of the sugar to adhere to the interior collection
surfaces of the evaporator; (b) providing an organic phase
comprising oil, a water miscible organic solvent, an active agent
dissolved in the solvent and a polymer or combination of polymers
for coating the oil core, wherein the active agent is selected from
the group consisting of: (a) a non-hydrophilic active agent and (b)
a hydrophilic active agent dissolved or suspended in an oil; (c)
slowly adding water to the organic phase to obtain an emulsion; (d)
continuously adding water to the emulsion to induce phase inversion
of the emulsion thereby obtaining an oil in water (o/w) emulsion;
(e) mixing the o/w emulsion with a gel forming polymer or a
combination of gel forming polymers; and (f) removing the organic
solvent by means of evaporation and water by means of spray drying
to obtain the spherical microcapsules.
[0012] In one embodiment, the present invention provides a
pharmaceutical dosage form comprising a composition and a
physiologically acceptable carrier, wherein the composition
comprises a plurality of microcapsules, each comprising a shell and
a core carrying an active agent selected from the group consisting
of: (a) a non-hydrophilic active agent and (b) a hydrophilic active
agent dissolved or suspended in an oil; wherein the shell comprises
a polymeric coating, and wherein at least a portion of the
microcapsules in a sample of the composition are spherical when the
sample of the microcapsules is viewed in a scanning electron
microscope with a magnification in the range of between .times.2000
and .times.50000.
[0013] In one embodiment, the present invention provides a method
of increasing bioavailability of an active agent in a human
subject's body; the method comprises administering to the human
subject a composition or a pharmaceutical dosage form comprising
the composition and a physiologically acceptable carrier, wherein
the composition comprises a plurality of microcapsules, each
comprising a shell and a core carrying an active agent selected
from the group consisting of: (a) a non-hydrophilic active agent
and (b) a hydrophilic active agent dissolved or suspended in an
oil; wherein the shell comprises a polymeric coating, and wherein
at least a portion of the microcapsules in a sample of the
composition are spherical when the sample of the microcapsules is
viewed in a scanning electron microscope with a magnification in
the range of between .times.2000 and .times.50000.
[0014] In one embodiment, the present invention provides a method
of treating a subject for a pathological condition which requires
for the treatment an effective blood level of an active agent, the
method comprises administering to the subject a composition or a
pharmaceutical dosage form comprising the composition and a
physiologically acceptable carrier, wherein the composition
comprises a plurality of microcapsules, each comprising a shell and
a core carrying an active agent selected from the group consisting
of: (a) a non-hydrophilic active agent and (b) a hydrophilic active
agent dissolved or suspended in an oil; wherein the shell comprises
a polymeric coating, and wherein at least a portion of the
microcapsules in a sample of the composition are spherical when the
sample of the microcapsules is viewed in a scanning electron
microscope with a magnification in the range of between .times.2000
and .times.50000.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 is a photograph of a small scale spray drying system
according to some embodiments of the present invention, employing
glass inner surfaces. The panel on the left of the figure is a
close-up view of the nozzle. Reference number I refers to the
beaker having a suspension of nanocapsules in the polymeric blend
ready to feed into the nozzle (infused via the dashed line by a
perilstatic pump), which is identified as Reference number III.
Reference number II refers to the cyclone for microcapsule
collection connected to the bottom of the collection flask.
Reference number III refers to the nozzle where hot air heats the
suspended feed and transforms the liquid to droplets transferred to
the drying chamber. The sugar which is spray dried prior to the
spray drying of the suspended polymeric blend adheres to the
surface of the cyclone and the collection flask.
[0016] FIGS. 2A-2E shows a series of scanning electron microscope
(SEM) images of microcapsules with irregular morphology containing
either the lipophilic active agent lopinavir (FIGS. 2A-2D), or a
hydrophilic synthetic peptide (FIG. 2E) made according to the
methods described in Example 1, at various magnifications
(.times.20000 (FIG. 2A), .times.12000 (FIG. 2B), .times.25000 (FIG.
2C) and .times.50000 (FIG. 2D), .times.3500 (FIG. 2E)).
[0017] FIG. 3 shows an SEM image of microcapsules with irregular
morphology containing lopinavir, at a magnification of .times.2000,
made according to the methods described in Example 2, wherein a
fine D-mannitol powder was added to the dry microcapsules following
their formation and collected from the spray dryer, hence this
D-mannitol was not spray dried.
[0018] FIGS. 4A-4C show SEM images of microcapsules containing a
non-hydrophilic or a hydrophilic drug in comparison to placebo
microcapsules made according to a method according to some
embodiments of the present invention. FIG. 4A is an SEM image of
symmetrical microcapsules bearing lopinavir loaded nanocapsules
prepared according to some embodiments of the present invention, in
which D-mannitol was spray-dried before the formation of the
microcapsules. FIG. 4B is an SEM image of symmetrical microcapsules
bearing peptide loaded nanocapsules prepared according to some
embodiments of the present invention in which D-mannitol was
spray-dried before the formation and collection of the
microcapsules. FIG. 4C is an SEM image of symmetrical microcapsules
bearing placebo nanocapsules without a drug prepared according to
some embodiments of the present invention, in which D-mannitol was
spray-dried before the formation and collection of the
microcapsules.
[0019] FIG. 5 shows the pharmacokinetic profiles of microcapsules
containing lopinavir made according to a method according to some
embodiments of the present invention. Two batches of microcapsules
without D-mannitol (B1 and B3) were compared to two batches of
microcapsules that were produced after the inner surfaces of the
evaporator were coated with dried D-mannitol according to some
embodiments of the present invention (B4 and B7).
[0020] FIGS. 6A and 6B show the internal and external morphology of
the microcapsules having a defined shell confirming a microcapsule
structure; hence they are not solid microspheres. FIG. 6A shows
nanocapsules released from microcapsules. FIGS. 6A and 6B confirm
the nano-in-micro formulation having a plurality of nanocapsules
entrapped in the inner space of a microcapsule.
[0021] FIG. 7 is an SEM image of mannitol particles as produced by
a spray drying of mannitol solution 1% Wt in water. This sample is
used as a control to distinguish between the morphology and size of
mannitol particles in comparison to the microcapsules as depicted
in FIGS. 4A, 4B and 4C. The mannitol sample of FIG. 7 was taken by
utilizing a high resolution electron microscope, which is based on
higher energy than the SEM used for FIGS. 4A-4C. The FIG. 7 shows
the mannitol particles as solid microspheres having symmetrical
morphology of size range from 1 to 10 .mu.m; which is the same size
range of the microcapsules. This microscopic image was produced
under controlled scanning electron microscopy conditions of
magnification .times.3000. It was found that mannitol microspheres
are not stable above this magnification, as they melt and loss
their solid texture above a magnification of .times.3000 while
under the same conditions the microcapsules of the invention are
stable. Hence, when a magnification above .times.3000 is used only
microcapsules are present in the sample. The relative instability
of mannitol microspheres at a magnification above .times.3000
enables differentiating between the symmetrical mannitol
microspheres and the symmetrical microcapsules.
[0022] FIG. 8 is a high resolution SEM image .times.2200 of a
nanocapsule in microcapsule formulation of the hydrophilic
polypeptide, exenatide. FIG. 8 depicts the symmetrical morphology
of the exenatide nano-in-micro formulation as an example for
hydrophilic macromolecules (polypeptide in comparison to the
example of the small peptide of FIG. 2E) and support the efficacy
of the mannitol procedure to improve the morphology of
microcapsules for both small peptides and polypeptide with high
molecular weight.
[0023] FIG. 9 is a high resolution SEM image of .times.55000 of a
single microcapsule and its surface morphology. In order to
distinguish between the appearance of mannitol solid microspheres
and the microcapsules in the SEM examples, a significant high
energy is applied to a single microcapsule to confirm its integrity
and stability. This example confirms the significant stability of
the microcapsules in comparison to the low stability of the
mannitol, which melts above the magnification energy of
.times.3000.
DETAILED DESCRIPTION
[0024] For clarity of disclosure, and not by way of limitation, the
detailed description of the invention is divided into the following
subsections that describe or illustrate certain features,
embodiments or applications of the present invention.
[0025] Further, as used herein, the term "comprising" is intended
to mean that the microcapsules include the recited elements, but
not excluding others. The term "consisting essentially of" is used
to define microcapsules that include the recited elements but
exclude other elements that may have an essential significance on
the bioavailability of the active agent within a subject's body.
"Consisting of" shall thus mean excluding more than trace elements
of other elements. Embodiments defined by each of these transition
terms are within the scope of this disclosure.
[0026] Further, all numerical values, e.g. when referring to the
amounts or ranges of the elements constituting the microspheres,
are approximations which are varied (+) or (-) by up to 20%, at
times by up to 10% from the stated values. It is to be understood,
even if not always explicitly stated, that all numerical
designations are preceded by the term "about".
Definitions
[0027] As used herein, the term "accommodated" refers to enclosing,
coating, embedding, surrounding, entrapping or any other manner of
incorporating the active agent by the gel forming polymer(s) so as
to provide a packed arrangement of the active agent with at least
the gel protecting environment.
[0028] The term "at least a portion of the spherical microcapsules"
as used therein refers to the portion that may be quantitatively
determined, e.g. by viewing the image of the micrograph, as well as
qualitatively determined, e.g. using image processing. "At least a
portion" may refer to at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 95%, 98%, 99% or even 100% of the microcapsules
being spherical at their outer surface.
[0029] The term "gel forming polymer" or "hydrogel forming polymer"
refers to hydrophilic polymer which when wetted, forms a network of
polymers that swell up or gels.
[0030] The term "nanocapsules" as used herein refers to nano- or
subnano-scale structures comprising an oil droplet (fine oil drops)
coated with a polymeric coating. The polymeric coating forms a hard
shell enveloping and forming the oil core.
[0031] As used herein, the term "non-hydrophilic active agent"
refers to any compound that is regarded as, at least to some
extent, water repelling. In other words, any agent exhibiting low,
medium or high hydrophobicity or lipophilicity would be regarded as
a non-hydrophilic agent. A non-hydrophilic agent may be defined by
parameters characterizing the partition/distribution coefficient of
the agent (as a solute) between two phases for example, an organic
solvent and water (the most commonly used system being
octanol-water). Typically, a partition coefficient (log P)
describes the hydrophobicity of neutral compounds, while the
distribution coefficient (log D, being a combination of pKa and log
P) is a measure of the pH dependent hydrophobicity of the agent. A
non-hydrophilic active agent in accordance with the invention is
any compound having a log P greater than 1.5.
[0032] As used herein, the term "plurality of microcapsules" refers
to two or more microcapsules accommodated in the composition.
[0033] As used herein, the term "plurality of nanocapsules" refers
to two or more nanocapsules accommodated in the gel-forming
polymer.
[0034] As used herein, the term "shell" refers to any solid or
semi-solid polymeric structure enclosing either, an oil droplet, or
a spherical microcapsule.
[0035] The term "spherical microcapsules" as used herein, refers to
micron- or submicron-scale particles which are typically composed
of solid or semi-solid materials and capable of carrying and
releasing a drug or any other active agent-containing nanocapsule
enclosed therein. When referring to spherical morphology it should
be understood as having its common meaning, i.e. a ball shaped,
globular outer structure/shape/morphology.
[0036] The term "sugar" as used herein refers to a simple sugar,
namely, mono, di or oligosaccharide or a sugar alcohol. Without
being limited, examples of simple sugars that can be employed in
the context of the present disclosure include inositole, lactose,
glucose, fructose, pentose, mannose and sucrose and any isomer
thereof.
[0037] The term "sugar alcohol" as used herein refers to a polyol,
such as that formed from a saccharide. Without being limited
thereto, a sugar alcohol may be any one of glycerol (3-carbon),
erythritol (4-carbon), threitol (4-carbon), arabitol (5-carbon),
xylitol (5-carbon), ribitol (5-carbon), mannitol (6-carbon),
sorbitol (6-carbon), galactitol (6-carbon), fucitol (6-carbon),
iditol (6-carbon, a cyclic sugar alcohol), volemitol (7-carbon),
isomalt (12-carbon), maltitol (12-carbon), lactitol
(12-carbon).
[0038] The term "water insoluble polymer" as used herein, refers to
any polymer which does not lose more than 10% of its dry weight
into an aqueous medium by dissolution, irrespective of the pH of
the medium.
[0039] The term "water soluble polymer" as used herein, refers to
any polymer which, when introduced into an aqueous phase at
25.degree. C., at a mass concentration equal to 1%, make it
possible to obtain a macroscopically homogeneous and transparent
solution, i.e. a solution that has a minimum light transmittance
value, at a wavelength equal to 500 nm, through a sample 0.1 cm
thick, of at least 80%, or alternatively, of at least 90%.
Preparation of Spherical Microcapsules
[0040] In some embodiments, the present invention provides
spherical microcapsules comprising a plurality of nanocapsules
situated within a lumen formed by the shell of the spherical
microcapsule, the nanocapsules comprising an oil core carrying an
active agent and a polymeric shell coating.
[0041] For illustrative purposes only, an example of a spherical
microcapsule according to some embodiments of the present invention
is shown in FIGS. 6A-6B. FIGS. 6A-6B show the internal and external
morphology of the microcapsules having a defined shell confirming a
microcapsule structure. FIGS. 6A and 6B confirm the nano-in-micro
formulation, having a plurality of nanocapsules entrapped in the
inner space of a microcapsule.
[0042] FIG. 9 is a high resolution SEM image of .times.55000 of a
single microcapsule and its surface morphology. Under high energy
conditions, of .times.15,000, no mannitol particles exist as such
particles start to melt above 3,000. Therefore, such high energy
conditions can distinguish between the mannitol and the
microcapsules.
[0043] In some embodiments, the spherical microcapsules are defined
as having a spherical symmetry where each point on the surface of
the spherical microcapsule has essentially the same distance from
the center.
[0044] In some embodiments, at least 5%, 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 95%, 98%, 99% or even 100% of the spherical
microcapsules have a spherical symmetry.
[0045] In some embodiments, the present invention provides a method
of preparing spherical microcapsules comprising a plurality of
nanocapsules situated within a lumen formed by the shell of the
spherical microcapsule, the nanocapsules comprising an oil core
carrying an active agent and a polymeric shell coating, the method
comprising: [0046] (a) spray drying sugar in a spray drying
evaporator, the amount of sugar being sufficiently in excess to
cause at least a portion of the sugar to adhere to the interior
collection surfaces of the evaporator; [0047] (b) spray drying a
dispersion comprising an active agent encapsulated in oil core
droplets of the plurality of nanocapsules and a gel forming polymer
or a combination of gel forming polymers to obtain microcapsules,
wherein the active agent is selected from the group consisting of:
(a) a non-hydrophilic active agent and (b) a hydrophilic active
agent dissolved or suspended in an oil, wherein a shell of the oil
core droplets comprises a polymeric coating; [0048] wherein at
least a portion of the microcapsules are spherical when viewed in a
scanning electron microscope with a magnification in the range of
between .times.20000 and .times.50000.
[0049] In some embodiments, the spray drying step (b) concentrates
the sugar on the inner surfaces of the evaporator.
[0050] In some embodiments, the inner surfaces of the evaporator
are glass. In some embodiments, the inner surfaces are stainless
steel.
[0051] In some embodiments, the present invention provides a method
of preparing spherical microcapsules comprising a plurality of
nanocapsules situated within a lumen formed by the shell of the
spherical microcapsule, the nanocapsules comprising an oil core
carrying an active agent and a polymeric shell coating, the method
comprising: [0052] (a) spray drying sugar in a spray drying
evaporator, the amount of sugar being sufficiently in excess to
cause at least a portion of the sugar to adhere to the interior
collection surfaces of the evaporator; [0053] (b) providing an
organic phase comprising oil, a water miscible organic solvent, an
active agent dissolved in the solvent and a polymer or combination
of polymers for coating the oil core, wherein the active agent is
selected from the group consisting of: (a) a non-hydrophilic active
agent and (b) a hydrophilic active agent suspended in an oil;
[0054] (c) slowly adding water to the organic phase to obtain an
emulsion; [0055] (d) continuously adding water to the emulsion to
induce phase inversion of the emulsion thereby obtaining an oil in
water (o/w) emulsion; [0056] (e) mixing the o/w emulsion with a gel
forming polymer or a combination of gel forming polymers; [0057]
(f) removing the organic solvent by means of evaporation and water
by means of spray drying to obtain the spherical microcapsules.
[0058] The organic solvent used can be any organic solvent miscible
with water that has a boiling point close to or lower than the
boiling point of water. A non-limiting list of such organic
solvents includes ethanol, methanol, acetone, ethyl acetate,
isopropanol (bp 108.degree. C., nonetheless regarded as volatile in
the context of the present disclosure).
[0059] The use of a combination of oil and organic solvent enables
the encapsulation within the nanocapsules of various active agents
which can be essentially non-hydrophilic in nature, or capable of
dissolving or being suspended in the oil core. The oil core can
also include one or more non-hydrophilic excipients (e.g.
lipophilic excipients). To this end, one or more excipients can be
added in the organic phase. The excipient can be any excipient
having at least 1% solubility in an oil phase. According to one
embodiment, the excipient is a lipophilic surfactant, such as
labrafil M 1944 CS, polysorbate 80, polysorbate 20.
[0060] Further, in accordance with this embodiment (where the agent
is encapsulated in nanocapsules). Water is slowly added (i.e. added
drop-wise) to the oil containing organic phase. At the beginning,
an oil in water emulsion is formed, i.e. drops of water are
suspended in the organic phase. However, continuous slow addition
of water to the medium eventually results in an inverse phenomenon,
where the continuous and non-continuous are `switched` such that
oil droplets coated with the polymer coating are suspended in
water. The continuous slow addition of water is stopped upon
achieving the inverse phenomenon (i.e. oil droplets coated with the
polymer coating suspended in water).
[0061] When forming the nanocapsules, initially a water in oil
(w/o) emulsion is formed and this w/o emulsion is converted to an
oil in water (o/w) emulsion by the addition of water to the
oil/organic phase.
[0062] The resulting emulsion comprising the oil droplets coated
with the coating polymer(s) is then mixed with the solution of the
gel forming polymer. Once the oil in water emulsion is formed and
the gel forming polymer is added, the solvent (or mixture of
solvents) and the water are essentially removed and the desired
composition comprising the spherical microcapsules is obtained.
[0063] Spray drying is a mechanical microencapsulation method
developed in the 1930s. Accordingly, an emulsion is atomized into a
spray of droplets by pumping the slurry through a rotating disc
into the heated compartment of a spray drying evaporator. There,
the solvent as well as the water in the emulsion are evaporated to
obtain the dry microcapsules.
[0064] The operation of the spray drying evaporator can vary
depending on the type of device used. In some embodiments, the
evaporator is operated under the following conditions:
[0065] Inlet temperature between 100.degree. C. to 200.degree. C.,
outlet temperature between 40.degree. C. to 100.degree. C. (the
outlet temperature being at least 50.degree. C. lower than the
inlet temperature, aspiration of 60% to 100%, pump rate in the
range of 10-40% that is 145-590 ml/h respectively and more
specifically 20-30%, air flow in the range of 300-800 L/h and more
specifically 600-700 L/h, and a nozzle cleaner range of 2-4. This
is the in-process cleaning mode of the nozzle mode -2 means
moderate rate of cleaning for spray drying of dispersions.
[0066] In some embodiments, the spray drying sugar comprises the
step of dissolving the sugar in water, e.g. double distilled water
(DDW) to obtain a solution of between 0.5%-10% w/v sugar, at times,
between 0.5% to 5%, at times 2%.+-.0.5%. The operating conditions
of the spray drying of the sugar solution are identical to the
spray drying of the spherical microcapsule dispersion despite the
inlet and outlet temperatures of inlet 100-115.degree. C. and
outlet 45-60.degree. C.
[0067] The existence of the dissolved sugar in the spray drying
components is evidenced by its appearance as a dried solid
particles/powder concentrated in the cyclone and the collection
flask.
[0068] As noted, subsequent to spray drying of the sugar solution a
dispersion of one or more gel forming polymers and the active agent
is subjected to spray drying in the same spray drying system a
priori "treated" with the sugar.
[0069] In some embodiments, the dispersion of the active agent with
polymers is prepared by dissolving the active agent with surfactant
in acetone followed by addition of the gel forming polymers in
water at the desired concentration of components.
[0070] In some embodiments, the active agent is first encapsulated
in nanostructures (e.g. nanocapsules).
[0071] In some embodiments, the composition comprises spherical
microcapsules where the active agent is enclosed within
nanocapsules. Such spherical microcapsules are spray dried as
described in U.S. Pat. No. 9,023,386, the content of which is
incorporated herein by reference, albeit in a spray dry evaporated
a priori treated with the sugar, as described herein.
[0072] Without being limited by theory, during the flow within the
spray drying system, most of the microcapsules are concentrated on
the interior collection surfaces of the cyclone and the collecting
flask and their relatively high affinity and surface area to the
interior collection surfaces leads to considerable adsorption of
the solid microcapsules to the glass, which in turn lead to the
undesired irregular morphology of the microcapsules.
[0073] However, unexpectedly, when evaluating the effect on the
morphology of microcapsules, with known additives such as
D-mannitol known as a bulk agent (used to improve the dispersion of
solid microparticles), it has been found that adding D-mannitol as
a raw powder or fine powder to the microcapsule powder, did not
affect the microcapsule and the bioavailability of this blend was
not improved. Similarly, manipulations in the spray drying
conditions, including changing the inlet and outlet temperature and
the air flow, neither affected microcapsule morphology, nor
bioavailability.
[0074] Spray-drying of D-mannitol solution before spray drying the
material forming the microcapsules; unexpectedly resulted in change
in morphology and improved bioavailability as further discussed
below.
[0075] Without being bound by theory, this unexpected finding was
postulated to obtain due to the adsorption of D-mannitol to the
interior collection surfaces that create a barrier between the
glass surface and the spray dried microcapsules, thereby reducing
the surface tension and the adsorption of the microcapsules to the
interior collection surfaces.
[0076] The spherical microcapsules according to some embodiments of
the present invention are comprised of aggregates of nanocapsules
incorporating (e.g. embedding, encapsulating, entrapping) the
active agent. Typically, the average diameter of the spherical
microcapsules according to some embodiments of the present
invention, which is understood as weight-average diameter as
determined by laser diffraction, ranges from approximately 10 .mu.m
to approximately 500 .mu.m. In some embodiments, the average
microsphere diameter is between about 10 .mu.m and about 20
.mu.m.
[0077] In some embodiments, the nanocapsules have an average
diameter of between about 100 nm and about 1000 nm. In some
embodiments, the nanocapsules have an average diameter of between
about 100 nm to 900. In some embodiments, the nanocapsules have an
average diameter of between about 100-300 nm to about 300-500
nm.
[0078] In some embodiments, the nanocapsules' size in a microsphere
is essentially uniform with about 99% of the oil droplets having a
diameter below 1 micron.
[0079] In some embodiments, the active agent is enclosed within the
nanocapsule. As a result, in some embodiments, there is no direct
contact between the active agent and the gel forming polymer
forming the spherical microcapsules.
[0080] In some embodiments, upon wetting and swelling in the GI
tract of the subject, the spherical microcapsules release the
nanocapsule and not the "naked" active agent.
[0081] In some embodiments, the present invention provides
pharmaceutical compositions comprising as the active component
spherical microcapsules comprising a plurality of nanocapsules
situated within a lumen formed by the shell of the spherical
microcapsule, the nanocapsules comprising an oil core carrying an
active agent and a polymeric shell coating.
[0082] In some embodiments, the oil core of the nanocapsules may
comprise a single oil type or a combination of oils and can be
selected from a wide range of usually usable oils from polar oils
to non-polar oils, as long as they do not mix with the water phase
and are a liquid as a whole in ambient and physiological
temperatures, ranging from about 20.degree. C. to about 37.degree.
C.
[0083] According to some embodiments, the oil droplets comprise an
oil selected from long chain vegetable oils, ester oils, higher
liquid alcohols, higher liquid fatty acids, natural fats and oils
and silicone oils.
[0084] According to some embodiments, the oil core comprises a
natural oil such as corn oil, peanut oil, coconut oil, castor oil,
sesame oil, soybean oil, perilla oil, sunflower oil, argan oil and
walnut oil.
[0085] In some embodiments, the oil droplets are each enclosed
within a polymeric coating to form nanocapsules comprising the oil
core and a polymeric shell surrounding the oil core.
[0086] In some embodiments, the polymeric coating provides a shell
structure surrounding the oil core. In some embodiments, the shell
can comprise a single polymer or a combination or blend of two or
more polymers. When the polymeric coating comprises a blend of
polymers, it is in accordance with some embodiments that at least
one of the polymers is soluble at a pH above 5.0, or that at least
one of the polymers is water soluble (pH independent).
[0087] In accordance with some embodiments, the combination of at
least two polymers comprises a blend of polymers comprising a first
polymer or polymers (group of polymers) which is either water
soluble (pH independent) or soluble at a pH of above 5.0 and a
second polymer or polymers (second group of polymers) which is
water insoluble polymer.
[0088] The term "polymer soluble at a pH above 5.0" refers to any
polymer that at a pH below 5.0 and at 25.degree. C., it does not
lose more than 10% of its dry weight into the medium by
dissolution, while at the same temperature, in an aqueous medium
having a pH above 5.0, it forms a hydrogel or dissolved to form a
macroscopically homogeneous and transparent solution. Such polymers
are referred to, at times; by the term "enteric polymers".
[0089] Water soluble polymers suitable for use in some embodiments
of the present invention include, but are not limited to, polyols
and polycarbohydrates. Examples include hydroxylated celluloses,
such as, for example, hydroxypropylmethyl cellulose and
hydroxymethyl cellulose. Other suitable water soluble polymers
include polyethylene glycol. Combinations of two or more water
soluble polymers are also contemplated.
[0090] Non-limiting examples of enteric polymers suitable for use
in some embodiments of the present invention include, but are not
limited to hydroxypropylmethylcellulose phthalate (HP55), cellulose
acetate phthalate, carboxy-methylcellulose phthalate, and any other
cellulose phthalate derivative, shellac, poly(methacrylic
acid-co-ethyl acrylate) 1:1 (Eudragit L100 55), zein.
[0091] In some embodiments, the enteric polymer is Eudragit L100
55.
[0092] Non-limiting examples of water insoluble polymers include
cellulose esters such as di- and triacylates including mixed esters
such as, for example, cellulose acetate, cellulose diacetate,
cellulose triacetate, cellulose propionate, cellulose acetate
butyrate, cellulose acetate propionate, cellulose tripropionate;
cellulose ethers such as ethyl cellulose; nylons; polycarbonates;
poly (dialkylsiloxanes); poly (methacrylic acid) esters; poly
(acrylic acid) esters; poly (phenylene oxides); poly (vinyl
alcohols); aromatic nitrogen-containing polymers; polymeric
epoxides; regenerated cellulose; membrane-forming materials
suitable for use in reverse osmosis or dialysis application; agar
acetate; amylose triacetate; beta glucan acetate; acetaldehyde
dimethyl acetate; cellulose acetate methyl carbamate; cellulose
acetate succinate; cellulose acetate dimethylamino acetate;
cellulose acetate ethyl carbonate; cellulose acetate chloroacetate;
cellulose acetate ethyl oxalate; cellulose acetate propionate; poly
(vinylmethylether) copolymers; cellulose acetate butyl sulfonate;
cellulose acetate octate; cellulose acetate laurate; cellulose
acetate p-toluene sulfonate; triacetate of locust gum bean;
hydroxylated ethylene-vinyl acetate; cellulose acetate butyrate;
wax or wax-like substances; fatty alcohols; hydrogenated vegetable
oils; polyesters, homo and copolymer, such as polylactic acid or
PLAGA and the like (for example poly(lactic-co-glycolic acid)
PLGA), and combinations thereof.
[0093] In some embodiments, the water insoluble polymers are
selected from the group consisting of: Poly(ethyl
acrylate-co-methyl methacrylate-co-trimethylammonioethyl
methacrylate chloride) 1:2:0.1 (Eudragit RS), Poly(ethyl
acrylate-co-methyl methacrylate-co-trimethylammonioethyl
methacrylate chloride) 1:2:0.2 (Eudragit RL), and combinations
thereof.
[0094] In some embodiments, the nanocapsules comprise at least two
polymers. In some embodiments, the first polymer is water insoluble
polymer and the second polymer is soluble at a pH above about
5.0.
[0095] In accordance with some embodiments, the weight/weight ratio
between the first polymer(s), i.e. the water insoluble polymer or
group of polymers and the second group polymer(s), i.e. the
polymer(s) soluble at pH above about 5.0 or group of such polymers
is in the range between 5:95 and 50:50.
[0096] Without being bound by theory, it is believed that the ratio
between the water insoluble polymer and the polymer soluble at pH
above about 5.0 (the "non-insoluble" polymer) is important for
controlling release of the active agent from the nanocapsules.
Without being bound by theory, having a first polymer that is water
insoluble and a second polymer that is soluble in water or soluble
in water at a pH above 5.0 allows, following exposure of the
nanocapsules to water or to an aqueous medium having pH above 5.0,
the slow dissolution of the polymer, while the general arrangement
of the insoluble polymer is essentially retained. In other words,
the slow dissolution of the "non-insoluble" polymer results in the
formation of channel-like pathways in a polymer "skeleton" formed
from the water insoluble polymer, through which the active agent
may escape the nanocapsule. In order to facilitate the controlled
release of the active agent from the nanocapsules, it has been
envisaged that one ratio between the first polymer, i.e. water
insoluble polymer, and the so-called "non-insoluble" polymer is
that in favor of the polymer soluble at a pH above about 5.0 (e.g.
a weight:weight ratio of 75:25 in favor of the water non-insoluble
polymer).
[0097] According to some embodiments, the polymeric combination
comprises a mixture of a first polymer or group of polymers (the
insoluble polymer) selected from Eudragit RL or Eudragit RS or a
combination of same, and a second polymer or group of polymers (the
water soluble or polymer soluble at a pH above 5.0) selected from
Eudragit L100 55 and hydroxypropyl methylcellulose phthalate
(HPMPC) or a combination of same. A specific selection of polymers
combination in accordance with the invention comprises Eudragit RS
and Eudragit L100-55 at a weight/weight ratio of from about 25:75
to about 50:50.
[0098] In some embodiments, the active agent is a non-hydrophilic
active agent. In accordance with some embodiments, the active agent
is any medicinal, cosmetic or diagnostic substance that, following
oral administration, its blood bioavailability is decreased or
inhibited as a result the P-gp efflux mechanism. P-gp substrates
may be categorized according to their solubility and level of
metabolism. A non-limiting list of P-gp substrates according to
this classification includes:
[0099] High solubility and extensive metabolism: amitryptyline,
cochicine, dexamethasone, diltiazem, ethinyl estradiol;
[0100] Low solubility and extensive metabolism: atorvastatin,
azithromycin, carbamazepine, cyclosporine, glyburide, haloperidol,
itraconazole, tacrolimus sirolimus, ritonavir, sanquinavir,
lovastatin.
[0101] High solubility and poor metabolism: amiloide, amoxicillin,
chloroquine, ciprofloxacin, dicloxacillin, erythromycin,
fexofenadine, levodopa, midazolam, morphine, nifedipine,
primaquine, promazine, promethazine, quinidine, quinine; and
[0102] Low solubility and poor metabolism--ciprofloxacin and
talinolol.
[0103] The active agent may be in free acid, free base or salt
form, and mixtures of active agents may be used.
[0104] In accordance with some embodiment, the active agent is a
lipophilic agent. The term "lipophilic agent" is used herein to
denote any compound that has a log P (octanol/water)>2.0-3.0 and
a triglyceride (TG) solubility, as measured, for example, by
solubility in soybean oil or similar, in excess of 10 mg/mL. This
definition includes medium lipophilic drugs i.e. having a log P
between 3.0 to 6, as well as highly lipophilic drugs, having a log
P greater than 6.
[0105] Examples of medium to lipophilic therapeutically active
agents which can be suitable for entrapment in the microcapsules
according to the present disclosure include the following:
[0106] Analgesics and anti-inflammatory agents: aloxiprin,
auranofin, azapropazone, benorylate, diflunisal, etodolac,
fenbufen, fenoprofen calcim, flurbiprofen, ibuprofen, indomethacin,
ketoprofen, meclofenamic acid, mefenamic acid, nabumetone,
naproxen, oxyphenbutazone, phenylbutazone, piroxicam, sulindac.
[0107] Anthelmintics: albendazole, bephenium hydroxynaphthoate,
cambendazole, dichlorophen, ivermectin, mebendazole, oxamniquine,
oxfendazole, oxantel embonate, praziquantel, pyrantel embonate,
thiabendazole.
[0108] Anti-arrhythmic agents: amiodarone, disopyramide, flecainide
acetate, quinidine sulphate.
[0109] Anti-bacterial agents: benethamine penicillin, cinoxacin,
ciprofloxacin, clarithromycin, clofazimine, cloxacillin,
demeclocycline, doxycycline, erythromycin, ethionamide, imipenem,
nalidixic acid, nitrofurantoin, rifampicin, spiramycin,
sulphabenzamide, sulphadoxine, sulphamerazine, sulphacetamide,
sulphadiazine, sulphafurazole, sulphamethoxazole, sulphapyridine,
tetracycline, trimethoprim.
[0110] Anti-coagulants: dicoumarol, dipyridamole, nicoumalone,
phenindione.
[0111] Anti-depressants: amoxapine, maprotiline, mianserin,
nortriptyline, trazodone, trimipramine maleate.
[0112] Anti-diabetics: acetohexamide, chlorpropamide,
glibenclamide, gliclazide, glipizide, tolazamide, tolbutamide.
[0113] Anti-epileptics: beclamide, carbamazepine, clonazepam,
ethotoin, methoin, methsuximide, methylphenobarbitone,
oxcarbazepine, paramethadione, phenacemide, phenobarbitone,
phenyloin, phensuximide, primidone, sulthiame, valproic acid.
[0114] Anti-fungal agents: amphotericin, butoconazole nitrate,
clotrimazole, econazole nitrate, fluconazole, flucytosine,
griseofulvin, itraconazole, ketoconazole, miconazole, natamycin,
nystatin, sulconazole nitrate, terbinafine, terconazole,
tioconazole, undecenoic acid.
[0115] Anti-gout agents: allopurinol, probenecid,
sulphin-pyrazone.
[0116] Anti-hypertensive agents: amlodipine, benidipine,
darodipine, dilitazem, diazoxide, felodipine, guanabenz acetate,
isradipine, minoxidil, nicardipine, nifedipine, nimodipine,
phenoxybenzamine, prazosin, reserpine, terazosin.
[0117] Anti-malarials: amodiaquine, chloroquine, chlorproguanil,
halofantrine, mefloquine, proguanil, pyrimethamine, quinine
sulphate.
[0118] Anti-migraine agents: dihydroergotamine mesylate, ergotamine
tartrate, methysergide maleate, pizotifen maleate, sumatriptan
succinate.
[0119] Anti-muscarinic agents: atropine, benzhexol, biperiden,
ethopropazine, hyoscyamine, mepenzolate bromide, oxyphencylcimine,
tropicamide.
[0120] Anti-neoplastic agents and Immunosuppressants:
aminoglutethimide, amsacrine, azathioprine, busulphan,
chlorambucil, cyclosporin, dacarbazine, estramustine, etoposide,
lomustine, melphalan, mercaptopurine, methotrexate, mitomycin,
mitotane, mitozantrone, procarbazine, tamoxifen citrate,
testolactone.
[0121] Tacrolimus and sirolimus.
[0122] Anti-protazoal agents: benznidazole, clioquinol,
decoquinate, diiodohydroxyquinoline, diloxanide furoate,
dinitolmide, furzoli done, metronidazole, nimorazole,
nitrofurazone, ornidazole, tinidazole.
[0123] Anti-thyroid agents: carbimazole, propylthiouracil.
[0124] Alixiolytic, sedatives, hypnotics and neuroleptics:
alprazolam, amylobarbitone, barbitone, bentazepam, bromazepam,
bromperidol, brotizolam, butobarbitone, carbromal,
chlordiazepoxide, chlormethiazole, chlorpromazine, clobazam,
clotiazepam, clozapine, diazepam, droperidol, ethinamate,
flunanisone, flunitrazepam, fluopromazine, flupenthixol decanoate,
fluphenazine decanoate, flurazepam, baloperidol, lorazepam,
lormetazepam, medazepam, meprobamate, methaqualone, midazolam,
nitrazepam, oxazepam, pentobarbitone, perphenazine pimozide,
prochlorperazine, sulpiride, temazepam, thioridazine, triazolam,
zopiclone.
[0125] beta-Blockers: acebutolol, alprenolol, atenolol, labetalol,
metoprolol, nadolol, oxprenolol, pindolol, propranolol.
[0126] Cardiac Inotropic agents: amrinone, digitoxin, digoxin,
enoximone, lanatoside C, medigoxin.
[0127] Corticosteroids: beclomethasone, betamethasone, budesonide,
cortisone acetate, desoxymethasone, dexamethasone, fludrocortisone
acetate, flunisolide, flucortolone, fluticasone propionate,
hydrocortisone, methylprednisolone, prednisolone, prednisone,
triamcinolone.
[0128] Diuretics: acetazolamide, amiloride, bendrofluazide,
bumetanide, chlorothiazide, chlorthalidone, ethacrynic acid,
frusemide, metolazone, spironolactone, triamterene.
[0129] Anti-parkinsonian agents: bromocriptine mesylate, lysuride
maleate.
[0130] Gastro-intestinal agents: bisacodyl, cimetidine, cisapride,
diphenoxylate, domperidone, famotidine, loperamide, mesalazine,
nizatidine, omeprazole, ondansetron, ranitidine,
sulphasalazine.
[0131] Histamine H,-Receptor Antagonists: acrivastine, astemizole,
cinnarizine, cyclizine, cyproheptadine, dimenhydrinate,
flunarizine, loratadine, meclozine, oxatomide, terfenadine.
[0132] Lipid regulating agents: bezafibrate, clofibrate,
fenofibrate, gemfibrozil, probucol.
[0133] Nitrates and other anti-anginal agents: amyl nitrate,
glyceryl trinitrate, isosorbide dinitrate, isosorbide mononitrate,
pentaerythritol tetranitrate.
[0134] Nutritional agents: betacarotene, vitamin A, vitamin
B.sub.2, vitamin D, vitamin E, vitamin K.
[0135] HIV protease inhibitors: Nelfinavir,
[0136] Opioid analgesics: codeine, dextropropyoxyphene,
diamorphine, dihydrocodeine, meptazinol, methadone, morphine,
nalbuphine, pentazocine.
[0137] Sex hormones: clomiphene citrate, danazol, ethinyl
estradiol, medroxyprogesterone acetate, mestranol,
methyltestosterone, norethisterone, norgestrel, estradiol,
conjugated oestrogens, progesterone, stanozolol, stibestrol,
testosterone, tibolone.
[0138] Stimulants: amphetamine, dexamphetamine, dexfenfluramine,
fenfluramine, mazindol.
[0139] Without being limited thereto, active agents in accordance
with the present disclosure include tacrolimus, sirolimus
halofantrine, ritonavir, lopinavir, amprenavir, saquinavir,
calcitrol, dronabinol, isotretinoin, tretinoin, risperidone base,
valproic acid while pro-drugs include dexamethasone palmitate,
paclitaxel palmitate, docetaxel palmitate.
[0140] Some non-limiting examples of lipophilic drugs which can be
incorporated into the composition disclosed herein and their
medical applications are described by Robert G. Strickley
[Strickley R. G. Pharmaceutical Research, 21(2):201-230; (2004)]
and by Kopparam Manjunath, et al. [Manjunath K. et al., Journal of
Controlled Release 107:215-228; (2005)].
[0141] In accordance with another embodiment, the active agent is
an amphipathic agent. The term "amphipathic agent" is used herein
to denote any compound that has a log P value between 1.5-2.5 and a
triglyceride (TG) solubility, as measured, for example, by
solubility in soybean oil or similar, in excess of 10 mg/mL.
[0142] Examples of amphipathic active agents which may be delivered
by the composition disclosed herein include, without being limited
thereto, pysostigmine salicylate, chlorpromazine, fluphenazine,
trifluoperazine, and lidocaine, bupivacaine, amphotericin B,
etoposide, teniposide and antifungal echinocandins and azoles, such
as clotrimazole and itaconazole.
[0143] Another example of a therapeutically, non-hydrophilic active
agent suitable for entrapment in the microcapsules disclosed herein
includes, without being limited thereto, is clozapine. Clozapine is
an effective atypical antipsychotic drug applied in the treatment
of resistant schizophrenia. Clozapine is rapidly absorbed orally
with a bioavailability of 27%. Clozapine is extensively metabolized
by hepatic microsomal enzymes (CYP1A2 and CYP3A4) and forms
N-demethyl and N-oxide metabolites.
[0144] Another example of an active agent suitable for entrapment
in the microcapsules disclosed herein includes, without being
limited thereto include any active agent that is soluble in the oil
core. Methods to determine the solubility of the active agent in
the oil core, and the composition of the oil core, are readily
determined by one of ordinary skill in the art. A person of
ordinary skill in the art would recognize that even hydrophilic
active agents can be incorporated into the oil core, provided the
hydrophiling agent is either soluble in an oil, or capable of being
modified to be soluble in an oil.
[0145] As a person versed in the art would understand, the
"hydrophilicity" of the materials is a characteristic of materials
exhibiting affinity for water, while the "hydrophobic" materials
possess the opposite response to water.
[0146] In some embodiments, the material hydrophobicity or
hydrophilicity may be due to the material's intrinsic behaviors
towards water, or may be achieved (or tuned) by modifying the
material by one or more of cross-linking the material,
derivatization of the material, charge induction to the material
(rendering it positively or negatively charged), complexing or
conjugating the material to another material and by any other means
known in the art.
[0147] Thus, in accordance with the present invention, the
selection of a material may be based on the material intrinsic
properties or based on the material's ability to undergo such
aforementioned modification to render it more or less hydrophobic
or hydrophilic. In some embodiments, the nanoparticle material
and/or the nanocarrier material may be cross-linked in order to
reduce material hydrophilicity (decrease solubility in aqueous
media).
[0148] In some embodiments, the active agent that is soluble in the
oil core is a peptide. In some embodiments, the solubility of the
peptide in a selection of oils is determined, and the oils in which
the peptide exhibits the greatest solubility is selected to
dissolve the peptide and form the spherical microcapsules according
to some embodiments of the present invention.
[0149] FIG. 8 is a high resolution SEM image .times.2200 of a
nanocapsule in microcapsule formulation of the hydrophilic
polypeptide, exenatide. Exenatide is a polypeptide with molecular
weight of 4186 Daltons which is significantly higher than the
hydrophilic peptide (panel E) which is 11924 Daltons. Therefore the
formulation of exenatide was based on the suspension (not
solubilization) of the polypeptide in the nanoencapsulation
procedure. As such; residual free exenatide (nonencapsulated in the
nanocapsules) remains in the microcapsules and therefore may affect
the microcapsules morphology.
[0150] In some embodiments, the oil core comprises the peptide or a
salt thereof, and one or more oil. In some embodiments, the oil
core is such that the peptide (or salt thereof) is solubilized
within the one or more oil, such that a homogenous formulation is
obtained.
[0151] In order to facilitate formation of nanocapsules of the
invention, the oil formulation may, in some embodiments, further
comprise at least one surfactant.
[0152] The term surfactant should be understood to encompass any
agent that is capable of lowering the surface tension of a liquid,
allowing for the formation of a homogeneous mixture of at least one
type of liquid with at least one other type of liquid, or between
at least one liquid and at least one solid. Thus, surfactants used
in the oil core of the invention may be used to control the surface
tension and surface interaction between the oil core and its
surroundings, thereby assisting in the process of forming the
nanocapsules.
[0153] Non-limiting examples of suitable surfactants are oleoyl
polyoxyl-6glycerides NF (Labrafil M1944 CS, Gatefosse) and all
nonionic oil surfactants exhibiting an HLP from 8 to 10.
[0154] In some embodiments, an organic phase is first obtained by
mixing the peptide-containing oil core with a solution of the
hydrophobic polymer in a solvent. The solvent, in some embodiments,
may be acetone.
Bioavailability
[0155] In some embodiments, the present invention provides a method
of increasing bioavailability of an active agent in a human
subject's body, the method comprising administering to the subject
spherical microcapsules, as described in this disclosure,
comprising a plurality of nanocapsules situated within a lumen
formed by the shell of the spherical microcapsule, the nanocapsules
comprising an oil core carrying an active agent and a polymeric
shell coating.
[0156] In some embodiments, the spherical microcapsules are
particularly useful for gastrointestinal delivery of active agents.
In some embodiments, the active agent is a substrate of P-gp efflux
pump. The term "substrate of the P-gp efflux pump" which may be
used interchangeably with the term "P-gp substrate" as used herein
refers to any active substance (for therapeutic, cosmetic or
diagnostic purposes) that is subject to active transport, "efflux"
out of cells via the P-gp membrane bound transporter. The P-gp is
expressed along the entire length of the gut and also in the liver,
kidney, blood brain barrier and placenta. In this context, the
present disclosure concerns medicinal substances subjected to
active transport by the intestinal P-gp which is located on the
apical membranes of the epithelial cells. Utilizing the energy that
is generated by hydrolysis of ATP, P-gp drives the efflux of
various substrates against a concentration gradient and thus reduce
their intracellular concentration and in the case of active
substances, their oral bioavailability.
[0157] Without being limited by theory, it has been found that the
resulting oral bioavailability of the encapsulated active agent
from the spherical microcapsules was significantly enhanced above
the encapsulated active agent in irregular microcapsules (not
symmetrically spherical) of the same composition (same type and
amount of active agent and polymers forming the microcapsules in
both formulations). The difference in morphology was evident from
scanning electron microscope at various magnifications, including
.times.20000.
[0158] In some embodiments, the improvement in bioavailability is
exhibited by enhanced absorption of the encapsulated active agent
from the gastrointestinal tract to the blood.
[0159] In some embodiments, the present invention provides a method
of treating a subject for a pathological condition. The method
comprises administering to the subject spherical microcapsules, as
described in this disclosure, comprising a plurality of
nanocapsules situated within a lumen formed by the shell of the
spherical microcapsule, the nanocapsules comprising an oil core
carrying an active agent and a polymeric shell coating wherein the
administration provides an amount of active agent sufficient to
treat the pathological condition.
[0160] The spherical microcapsules, according to some embodiments,
may be formulated in accordance with any desired application. There
are almost limitless applications for such spherical
microencapsulated material. Depending inter alia, on the active
agent, the composition may be applicable in agriculture,
pharmaceuticals, foods, cosmetics and fragrances, textiles, paper,
paints, coatings and adhesives, printing applications, and many
other industries.
[0161] In accordance with some embodiments, the composition is for
use in medicine, cosmetics or diagnosis.
[0162] In yet some further embodiments, the composition is
formulated as a pharmaceutical dosage form, the dosage form
comprising, in addition to the composition a physiologically
acceptable carrier.
[0163] In some embodiments, the physiologically acceptable carrier
is suitable for oral administration. To this end, the dry
composition may be included in an enteric vehicle, such as an
enteric capsule. Non-limiting examples of enteric capsules include
soft or hard entero-coated capsules as known in the art.
[0164] For oral delivery tablets, pills, powders, lozenges,
sachets, cachets, elixirs, suspensions, aerosols (as a solid or in
a liquid medium), and sterile packaged powders as well as other
delivery forms can be used.
[0165] Compositions comprising spherical microcapsules according to
some embodiments of the present invention were shown to provide
elevated blood levels of the active agents exemplified as compared
to microcapsules having irregular shape, i.e. produced by the same
step, albeit without a prior spray drying the bulk agent (e.g.
sugar) in the evaporator.
[0166] In yet some embodiments, the pharmaceutical dosage form is
formulated for administration by injection.
[0167] According to some embodiments, the pharmaceutical dosage
form disclosed herein provides controlled release of the active
agent. As used herein, "controlled release" means any type of
release which is not immediate release. For example, controlled
release can be designed as modified, extended, sustained, delayed,
prolonged, or constant (i.e. zero-order) release. In theory, one of
the most useful release profiles is constant release over a
predetermined period of time.
[0168] The present disclosure also provides a method of increasing
bioavailability of an active agent in a human subject's body, the
method comprises providing the subject with the composition
disclosed herein (comprising spherical microcapsules). The results
presented herein show that, by the use of the spherical
microcapsule, bioavailability of the tested active agents in the
blood increases, at least by a factor of 2, with respect to control
irregularly shaped microcapsules (See FIG. 5).
[0169] The present disclosure also provides a method of treating a
subject for a pathological condition which requires for the
treatment an effective amount of an active agent within the
subject's blood system, the method comprises providing the subject
with the composition disclosed herein.
[0170] The term "pathological condition" used herein denotes any
condition which requires for improving the well-being of the
subject the delivery, of an active agent being a drug or pro-drug
or diagnostic agent, such as those listed hereinabove.
[0171] In some embodiments, when the active agent is a
non-hydrophilic entity, such as, without being limited thereto, a
lipophilic agent, or any lipophilic derivative of an active agent,
the delivery of the active agent in accordance with the present
disclosure is via the intestines. In some embodiments, the delivery
of the active agent is via the intestinal paracellular,
trancellular or lymphatic transport, or a combination thereof. The
non-limiting list of conditions includes, inter alia, inflammation
and autoimmune disorders, parasitism (e.g. malaria) bacterial,
viral or fungal infection, cardiac disorders (e.g. arrhythmia),
coagulation disorders, depression, diabetics, epilepsy, migraine,
cancer, immune disorders, hormonal disorders, psychiatric
conditions, gastrointestinal tract disorders, nutritional
disorders, and many others, as known in the art.
[0172] In some embodiments, when the active agent is a hydrophilic
active agent dissolved or suspended in the oil core, the delivery
of the active agent in accordance with the present disclosure is
via the intestines. In some embodiments, the delivery of the active
agent is via the intestinal paracellular, trancellular or lymphatic
transport, or a combination thereof. The non-limiting list of
conditions includes, inter alia, inflammation and autoimmune
disorders, parasitism (e.g. malaria) bacterial, viral or fungal
infection, cardiac disorders (e.g. arrhythmia), coagulation
disorders, depression, diabetics, epilepsy, migraine, cancer,
immune disorders, hormonal disorders, psychiatric conditions,
gastrointestinal tract disorders, nutritional disorders, and many
others, as known in the art.
[0173] The effective amount of active agent in the pharmaceutical
dosage form can vary or be adjusted widely depending upon the
particular application, the manner or introduction, the potency of
the particular active agent, the loading (or not) of the agent into
nanocapsules, and the desired concentration. The effective amount
is typically determined in appropriately designed clinical trials
(dose range studies) and the person versed in the art will know how
to properly conduct such trials in order to determine the effective
amount. As generally known, an effective amount depends on a
variety of factors including the hydrophobicity of the active agent
and when relevant, the lipophilicity, the selection of polymers
forming the nanocapsule (the oil droplet's coating) and/or the
outer gel forming envelop, the distribution profile of the active
agent within the body, a variety of pharmacological parameters such
as half-life in the body, on undesired side effects, if any, on
factors such as age and gender, etc.
[0174] The pharmaceutical dosage form can be administered over an
extended period of time in a single daily dose, in several doses a
day, as a single dose and in several days, etc. The treatment
period will generally have a length proportional to the length of
the disease process and the specific microsphere's effectiveness
(e.g. effective delivery via the intestines, effectiveness of the
agent etc.) and the patient species being treated.
[0175] The present invention is further illustrated, but not
limited by, the following examples.
EXAMPLES
[0176] As will be shown in the following non-limiting examples, the
effect of the spherical morphology (vs. the irregular morphology
obtained without the addition of the bulk agent, e.g. D-mannitol)
was evaluated. More specifically, the pharmacokinetics of the
active agent accommodated in the microcapsules (irregular shape vs.
spherical shape) following oral administration to rats was
examined. The assumption, based on hitherto known art was that the
spherical microcapsules will reduce the absorption of the
encapsulated active agent via the gastrointestinal tract due to
their reduced surface area in comparison to irregular surfaces. The
concern was that with the decreased surface area of the spherical
microcapsules, the interaction between the surfaces of the
microcapsules with the gastrointestinal mucosa will be reduced as
well. Surprisingly, it was found that the uniform and spherical
microcapsules exhibited a significant increase of oral
bioavailability much above (at least twice) that of the irregular
microcapsules.
[0177] One of ordinary skill in the art can readily appreciate that
the process disclosed below, using a small-scale evaporator can be
scaled up to a larger scale, or industrial scale.
Materials and Methods
Materials
[0178] D-Mannitol EP, FCC, USP was purchased from Sigma Aldrich.
PLGA (Lactel end capped hexandiol 50:50 intrinsic viscosity
0.15-0.25 dL/g, lot No. A11-045, expiry date May 17, 2015);
[0179] Poly(methacrylic acid), Ethyl acrylate 1:1 (Eudragit.RTM.
L100-55) was obtained from Evonik Rohm (Kirschenallee,
Germany);
[0180] Hydroxypropylmethylcellulose (Methocel E4M Premium) was
purchased from Dow Chemical Company (Midland, Mich., USA);
[0181] Oliec acid was purchased from Fisher Scientific UK. Octanoic
acid was purchased from Merck KGaA (Germany);
[0182] Sodium phosphate monobasic, monohydrate was purchased from
Mallinckrodt Chemicals (Phillipsburg, N.J., USA);
[0183] Phosphate buffered saline was obtained from Biological
Industries (Kibbutz Beit Haemek, Israel);
[0184] All organic solvents were HPLC grade and purchased from J.T.
Baker (Deventer, Holland);
[0185] Lopinavir (99.6% purity) pharma grade API (Active
Pharmaceutical Ingridient) was purchased from Aurobindo Pharma Ltd.
India.
Techniques
[0186] The nanocapsules within microcapsules were prepared using a
spray drying technique. FIG. 1 is an image of a spray drying system
used in accordance with this example (Buchi mini-sprayer dryer
Model B-290), showing specifically the beaker (I) containing a
suspension of nanocapsules (NC) in a polymeric solution, the tube
directing the suspension into the nozzle (dashed arrow), and the
cyclone (II) where the spray dried powder is collected and the
nozzle (III) from which the suspension is spray dried.
Methods
[0187] In all examples below microcapsules contained nanocapsules
prepared as described below:
Nanocapsule (NC) Preparation
[0188] The primary NCs were first prepared by dissolving 1500 mg OA
(oleic acid), 300 mg labrafil M 1944 CS, 300 mg PLGA and 450 mg LPV
in 300 ml of acetone. Then, 210 ml of water (Double Distilled
Water--DDW) were added slowly to the oil phase, creating an o/w
emulsion, as evidenced by the rapid formation of opalescence in the
suspension medium.
[0189] Separately, the NCs were prepared by dissolving in 100 mL of
acetone: 300 mg of PLGA (Lactel end capped hexandiol 50:50
intrinsic viscosity 0.15-0.25 dL/g). In separate glass vials, 25 mg
of an eight amino acid peptide was dissolved in 120 mg of octanoic
acid with vortex for 5 minutes at room temperature. To the same
vial, 380 mg of oleic acid and 100 mg of Labrafil M 1944 CS were
added and then vortexed vigorously for 2 minutes until complete
dissolution. The contents of the vial were transferred to the
acetone solution and washed three times with acetone to confirm
that all the oil solution was added to the acetone-PLGA solution.
70 mL of bi-distilled water were slowly added to the organic phase
while stirring at 1000 rpm until an O/W (oil-in-water) suspension
formed (NCs suspension).
Example 1: Preparation of Irregular Microcapsules
[0190] Microcapsules were formed by microencapsulating either
lopinavir or an eight amino acid peptide or a polypeptide of 4168
dalton which was encapsulated in nanocapsules (NCs) using the
spray-drying technique using a system as shown in FIG. 1.
[0191] Specifically, 132.26 mg of NaH.sub.2PO.sub.4.H.sub.2O were
dissolved in 150 ml of doubled distilled water (DDW) and pH was
adjusted to 6.5 using NaOH 1N. To this solution, 750 mg of Eudragit
L 100-55 were added and pH was adjusted again in the same manner to
6.5. Then 100 ml of HPMC solution were prepared by first dispersing
1 g of HPMC in 100 ml of DDW at about 80.degree. C., and then
cooled to room temperature under stirring to dissolution. The
Eudragit L 100-55 at pH of 6.5 was added to HPMC solution and the
combined mixture of Eudragit+HPMC was poured via funnel to the NCs
dispersion medium at room temperature. The combined mixture of NCs
with the polymer solution of HPMC+Eudragit was transfer to rotor
evaporator and the acetone was evaporated under room temperature.
At the end of acetone evaporation an equal volume (by weight) of
DDW was added to obtain total volume of 760 g.
[0192] The morphology of the reference formulations is depicted in
FIGS. 2A-2E, evidencing their irregular morphology. Microcapsules
containing lopinavir are shown in FIGS. 2A-D. Microcapsules
containing an eight amino acid peptide are shown in FIG. 2E.
Example 2: Preparation of Spherical Microcapsules
[0193] The spherical microcapsules according to the present
disclosure were prepared in combination with D-mannitol.
Specifically, Spray drying of D-mannitol was performed prior to the
spray drying of the material for forming the NCs/microcapsules
polymers suspension as described in Example 1. FIG. 7 is an SEM
image of mannitol particles used as a control to distinguish
between the morphology and size of mannitol particles in comparison
to the microcapsules as depicted in FIGS. 4A, 4B and 4C. The
rationale was to reduce the inherent surface energy of the interior
surfaces of the cyclone and the receiver flask and by that to
prevent an immediate adsorption of the dry microcapsules to the
interior collection surfaces. Without being bound by theory, it was
assumed that the irregular morphology of the microcapsules (without
mannitol) is mainly due to their adsorption to the interior
collection surfaces during their spray drying. Therefore, a
D-mannitol solution of 1% w/v in DDW (100 ml) was spray dried by a
Buchi mini spray-drier B-290 apparatus (Flawil, Switzerland) under
the following conditions: Inlet temperature 110.degree. C.; outlet
temperature 50.degree. C.; aspiration 100%; pump rate 20% and
nozzle cleaner 2. The dried D-mannitol powder was
accumulated/adsorbed on the surface of the cyclone separator and
the receiver flask surfaces. The morphology of the innovative
formulation is depicted in FIG. 3.
Example 3: Preparation of Mannitol Fine Powder--Microcapsule
Combination (Control Formulation)
[0194] In order to show that the effect of mannitol on the
morphology of microcapsules is specific to spray drying procedure
and has no effect on the delivery of the drug per se, a fine powder
of mannitol (grinned by pastel and mortar) was added and mixed with
a sample of the irregular MC (prepared according to Example 1). The
irregular MC was premixed for 3 hours (h) by continuous slow
magnetic stirring at 200 rpm. The morphology of this mixed sample
is shown in FIGS. 4A-4C.
Example 4: Spherical Microcapsules
[0195] Encapsulation of NCs in microcapsules was obtained by the
spray drying of the NCs in HPMC/Eudragit polymers blend as
described in Example 1. However, the suspension was spray-dried
immediately after the spray drying of mannitol under the following
conditions: Inlet temperature 160.degree. C.; outlet temperature
98.degree. C.; aspiration 100%, pump rate 20% (the feeding rate was
4 ml/min) and nozzle cleaner 2. The powder of the microcapsules
with D-mannitol was accumulated in the cyclone separator and
collected later. The average yield of the process was 50-70%.
Physicochemical Characterization of the Spherical Microcapsules
Drug Content in the Final Dried Microcapsule Formulations
[0196] Samples of 10 mg were taken from the formulation. The
samples were completely dissolved in 3 ml DMSO, in a volumetric
flask under agitation for 1 h and the volume was then adjusted to
10 ml with methanol. 975 .mu.l were withdrawn from each flask and
the volume was completed to 1000 .mu.l with internal standard (10
.mu.g) solution of diazepam in methanol. Finally, for all
formulations, the drug content was determined by injecting 20 .mu.l
from each sample into an HPLC device, under the following
conditions: Mobile phase--ACN:H2O 45:55, flow rate--0.8 ml/min,
wavelength--210 nm, column--XTerra MS C8 5 .mu.m 3.9.times.150 mm
purchased from Waters (Milford, Mass., USA, distributed by
Medtechnica. Petach-Tikva, Israel). Two calibration curves were
constructed from LPV concentrations ranging between 0 to 200
.mu.g/ml and internal standard diazepam at a concentration of 10
.mu.g/ml. The samples of the first calibration curve were dissolved
in methanol and the samples of the second curve were dissolved in
DMSO:MeOH 3:7 as were the formulation samples. The calculated
recovery percentage was 100%.
Mannitol Content of the Final Dried Microcapsule Formulations
[0197] Mannitol content is assayed by commercial colorimetric assay
kits as an example D-mannitol colorimetric assay kit--MAK096 Sigma
Aldrich. Briefly: D-Mannitol Colorimetric Assay kit provides a
simple and direct procedure for measuring D-mannitol in a variety
of samples, including fruit and juices. D-Mannitol concentration is
determined by a coupled enzyme assay, which results in a
colorimetric (450 nm) product proportional to the D-mannitol
present. The sugar alcohol L-arabitol also acts as a substrate for
this assay. Therefore, this kit can be used to measure
L-arabitol.
Morphological Evaluation--SEM
[0198] Morphological evaluation of spray-dried NC-loaded
microspheres was carried out using High-Resolution Scanning
Electron Microscope (Sirion, HR-SEM; FEI Company, The Netherlands).
The specimens were fixed on an SEM-stub using double-sided adhesive
carbon tape or alternatively.
[0199] The SEM micrograph (magnification .times.2000) of
microcapsules bearing a drug (Lopinavir) loaded NCs as a result
from their spray-drying after spray drying of D-mannitol (1% w/v in
water) is provided in FIG. 3. As observed the microcapsules plus
mannitol have a well-defined symmetrical spherical morphology.
[0200] In comparison, irregular microcapsules were prepared without
the D-mannitol (in accordance with the procedure of U.S. Pat. No.
9,023,386). Typical SEM micrographs of such Reference microcapsules
are shown in FIGS. 2A-2E (magnifications .times.20000,
.times.12000, .times.25000, .times.50000, and .times.3500,
respectively). FIGS. 2A-2E depict the irregular morphology of the
spray-dried microcapsules of two independent batches at various
magnifications.
[0201] In addition, FIG. 4A-4C shows SEM micrograph of the
irregular microcapsules with D-mannitol added after their
production as fine powder to the microcapsule powder. The
D-mannitol crystals are observed near the irregular microcapsules.
Hence, the mixture of D-mannitol fine powder with the solid spray
dried microcapsules didn't modify the morphology of the spray dried
irregular microcapsules.
In Vivo Studies--Pharmacokinetic Studies in Rats
[0202] All the animal studies in this research were carried out in
accordance with the rules and guidelines concerning the care and
use of laboratory animals and were approved by the Israeli ethical
committee.
[0203] Sprague Dawley male rats weighing 300-320 g were used in
this study and were separated randomly into to evaluate the biofate
of LPV in different formulations. The animals were housed in SPF
conditions, fasted overnight (12-14 h prior to the experiment) with
free access to water. The animals were given a dose of 10 mg/kg
LPV. All the microparticulate formulations, I prepared by
dispersing the NCs embedded in microparticles in DDW to the final
concentration of 32 mg/ml. Blood samples (400-500 .mu.l) were
withdrawn from the tail vein at 0, 0.5, 1, 2, 4, 8, 12 and 24 h.
Saline solution was given to the animals after 30 min and then
again after 3 h. The blood samples were collected in heparin
containing tubes. The samples were immediately centrifuged at
10,000 rpm for 5 min, after which 200-300 .mu.l of plasma samples
were transferred to new tubes and stored at -80.degree. C. until
further analysis by LC-MS/MS as described below.
[0204] The blood samples were treated by protein precipitation in
methanol, following 15 min centrifuge at 10,000 rpm. 25 ng of
quinoxaline (QX) dissolved in methanol were added to each sample,
as an internal standard. The supernatant layer was collected and
the samples were injected into an LC-MS/MS device under the
following conditions: A Phenomenex Kenetex, column (RP-C18,
50.times.2.1 mm, 2.6 .mu.m, 100A) in gradient mode. The mobile
phase consisted of A=methanol/formic acid 99.9/0.1 and
B=water/formic acid 99.9/0.1. The A/B ratio is 58/42 at t=0 min,
during the first 1.5 min the ratio changed gradually to A/B 80/20
and remained steady until 2.1 min. The ratio changed again to A/B
58/42 rapidly from 2.1-2.15 min and remained constant until the end
of the run at 5 min, for the purpose of system cleaning and
stabilization. The flow rate was maintained at 0.35 ml/min, and the
column temperature was maintained at 35.degree. C. LC-MS/MS
analysis was performed with a thermo scientific Accela HPLC system
coupled with a TSQ Quantum Access MAX detector in positive
ionization mode. Detection and quantification were carried out by
multiple-reaction monitoring with transitions from m/z 629.3 to 156
for LPV and from m/z 313.1 to 246 for QX. The tested samples were
quantified against a calibration curve in the range of 0-100 ng/ml.
The correlation coefficient values were higher than 0.99 indicating
that good linearity, accuracy and specificity were achieved.
[0205] The comparative pharmacokinetics profiles of the spray dried
microcapsules with and without D-mannitol (irregular and spherical
morphology respectively) is provided in FIG. 5. Specifically, the
microcapsule powder was suspended in water and administered to rats
by gavage by equal dose of 10 mg/kg of the encapsulated drug
lopinavir. Two batches of microcapsules without D-mannitol (B1 and
B3) were compared to two batches of microcapsules with spray dried
D mannitol (B4 and B7), the formulation of the NCs and the spray
drying conditions (inlet and outlet temperatures and air flow) were
the same for all batches. The only variable is the spray drying of
D-mannitol before the spray drying of the microcapsules (in batches
B4 and B7). The formulations B1 and B3 were produced without the
pre-spray-drying of Mannitol, whereas B4 and B7 included the
pre-spray-drying of mannitol. FIG. 5 shows that plasma levels of
the MC with D-mannitol is much higher as compared to the blood
level of the irregular MC (prepared without prior spraying with
D-mannitol).
[0206] The comparative AUC (area under the plasma lopinavir time
curve) values of the tested groups as for extent of absorption is
provided in Table 1 below.
TABLE-US-00001 TABLE 1 AUC Batch AUC B7 (n = 5) 2452 B4 (n = 3)
2104 B1 (n = 3) 1247 B3 (n = 3) 883
[0207] The microcapsules with the prior spray drying of D-mannitol
doubled the extent of absorption above the microcapsules with the
irregular morphology (without D-mannitol). The increase of Cmax was
significantly higher in B4 and B7 in comparison to B1 and 3 as
revealed by the paired t-test, p<0.05 as shown in Table 2
below.
TABLE-US-00002 TABLE 2 P values (t-test) Time P values point (h)
B7/B1 P values B7/B3 P values B4/B1 P values B4/B3 1 0.012067
0.003691 0.276124 0.009849 2 0.487524 0.215681 0.032535 0.008087 4
0.267686 0.159417 0.038736 0.079601 6 0.2363 0.092573 0.238343
0.134816
* * * * *